Liquid-Liquid Extraction

• The separation of a component from a liquid mixture by

treatment with a solvent in which the desired component is
preferentially soluble is known as liquid-liquid extraction.
• Since this is the operation between the two liquid phases, no
vaporisation is needed; thus, extraction can be performed at low
temperatures.
• Accordingly, extraction is suitable for separating materials that
may decompose or denature at elevated temperatures.
• Examples of the uses of extraction are:
― The separation of penicillin from the broth (the liquid phase
obtained from biological processes)
― The separation of aromatic-ring hydro-carbons (e.g.,
benzene, toluene) from paraffins using sulpholane
Generally, the solvent should:
• be able to dissolve the solute more than the diluent
• be immiscible to the diluent
• be highly selective for the desired solute
• not be selective for contaminants
• non-toxic/ non-corrosive
• non-reactive or chemically stable
• non-flammable or non-explosive
• readily available/ inexpensive
• environmentally friendly (i.e. “green”)
• High distribution coefficient: KD=YL/XH
YL and XH are concentrations of the solute in Light, YL

light and heavy phases, respectively.

Heavy, XH
The light phase = organic solvent
The heavy phase = fermentation broth
• The simplest liquid-liquid extraction involves only a ternary (i.e. three
components) system.
• The solution which is to be extracted is called the feed, and the liquid
with which the feed is contacted is the solvent.
• The solvent-rich product of the operation is called the extract, and the
residual liquid from which solutes has been removed is the raffinate.
 Types of Extractor
• Single Stage • Multi Stage
 Application of Extraction
• Extraction processes are well suited to the petroleum
industry because of the need to separate heat-
sensitive liquid feeds according to chemical type (e.g.
aliphatic, aromatic, naphthenic) rather than by
molecular weight or vapour pressure.
• Other major applications exist in the biochemical or
pharmaceutical industry, where emphasis is on the
separation of antibiotics and protein recovery.
• In the inorganic chemical industry, they are used to
recover high-boiling components such as phosphoric
acid, boric acid, and sodium hydroxide from aqueous
solutions.
 Protein Precipitation
Protein precipitation is widely used in downstream processing of
biological products in order to concentrate proteins and purify
them from various contaminants.
 Protein Precipitation by Salt
• Proteins show a variation in solubility depending on
the ionic environment of their solution.
• When low concentrations of salt is added to a protein
solution the solubility increases.
• Beyond a certain point after continuing addition of
salt, the protein solubility start to decrease leading to
exclusion of protein out of the solution in the form of
precipitate.
• The point where the precipitation start is different
between different protein.
 Salting In
• Low concentrations of salt the solubility increases.
• Salt molecules stabilize protein molecules by
decreasing the electrostatic energy between the
protein molecules which increase the solubility of
proteins.

-
H2O
H2O
+
H2O
Salt H2O
+
+ H2O
H2O
-
 Salting out
• High concentration of salts solubility decreases, and protein
precipitates.
• because the excess ions (not bound to the protein) compete with
proteins for the solvent.
• The decrease in solvation allows the proteins to aggregate and
precipitate. The protein molecules tend to associate with each
other because protein-protein interactions become energetically
more favorable than protein-solvent interaction.
- -
- +
H2O + H2O
H2O
+
H2O More salt + +
+
-
+ H2O - +
- - -
H2O - + H2O
+ + -
 Salting Out

• Unwanted proteins can be

removed from a protein
solution mixture by salting
out
• After removing the
precipitate by filtration or
centrifugation, the desired
protein can be precipitated by
altering the salt
concentration to the level at
which the desired protein
becomes insoluble.
• Salting out with ammonium sulfate is a technique that
is used as an early step in purification scheme.
• Significant purification is not achieved, but broad
ammonium sulfate cut at least a volume of unwanted
proteins.
• The amount of salt needed to isolate a specific protein
is determined from the salt's fractionation table.

Purification
of LS-24
from seeds
of L. sativus

Qureshi et al., 2006

Ammonium Sulfate Fractionation for Protein Purification
 Precipitation with Organic Solvents
• Polar organic solvents such as aliphatic alcohols (ethanol) and
ketones (acetone) reduce protein solubility.
• Decreasing surface hydration and increasing hydrophobic
surface promoting aggregation like salt effect.
• However, such effects are less involved because of solubilizing
influence of organic solvent on these areas.
• Protein denaturation is the major drawback to this approach
and is largely avoided by using polyalcohols.
• Efficient precipitation method for cytoplasmic and other water
soluble proteins, less efficient for hydrophobic membrane
proteins.
The size of protein molecule is an important factor for aggrgation;
the larger the molecule, the lower the percentage of organic
solvent required to precipitate it.

6.0
phosphorylase
Log molecular weight

pyruvate kinase

lactate dehydrogenase
5.0
enolase

phosphoglycerate kinase

myokinase

parvalbumin
4.0
0 20 40 60 80

Acetone concentration (% vol/vol)

 Heat/pH-induced Precipitation
• The precipitation strategy involve the option to use heat or pH
to denature and precipitate the unwanted proteins, while the
desired protein remains unaffected.
• Known as subtractive adjunct precipitation.
• Some proteins such as adenylate kinase, plant protein inhibitors,
trypsin and certain proteins of thermophillic organisms are
relatively heat stable.
• Similarly some proteins are biologically active outside normal pH
range of 5 – 10.
• This approach is now commonly used for single-step purification
of thermo-stable enzymes expressed in mesophilic host.
• Proteins differ in their thermal stability and ability to renature
after thermal denaturation.
• Calmodulin is an excellent example of a protein that can be
purified by thermal denaturation.

% Native structure calmodulin

Average
protein

0 100
Temp, C

• In general, smaller and highly charged proteins are stable to

higher temperatures than large and more hydrophobic proteins.
• A major limitation to the use of this technique is the action of
proteases which are inherently resistant to denaturation.
 Precipitation with Polymers
• Nonionic water soluble polymers.
• Several nonionic water soluble polymers cause precipitation of
proteins, however, high viscosity of many of them make their
use rather difficult.
• Polyethylene glycol (PEG), one exception which can be used
thoroughly because upto 20% (w/v) of this solution is not
viscous.
• Available in variety of degrees of polymerization
H-(CH2-CH2-O)n-H polymer of ethylene oxide
• High molecular weight called polyethylene oxide (PEO) and low
molecular weight called polyethylene glycol (PEG).
• PEG precipitation is somewhat similar to organic solvent
precipitation, and PEG molecules are regarded as polymerized
organic solvent.
• PEG precipitation is quite selective for fractionating
plasma/serum proteins, so has found wide application in clinical
diagnostic testing e.g., prolactin, protein S measurements.
• PEG with an average molecular weight of 4000 – 6000 is
commonly used.
• Very mild and selective method, less tendency to denature
proteins.
• Disadvantge is that PEG is not easily removed from the protein
fraction.
• However, polymer can be removed in subsequent stages of
purification scheme.
Examples of PEG Precipitations:

1. Thyroid stimulating immunoglobulins

2. Fractionating Collagen types

3. Acid phosphatases

4. Lactoglobulins

5. Lipases

6. Nucleic acid precipitations and purifications

PEG Precipitations
Advantages
Precipitation with polymers retains more intact
structure of the proteins as compared to other
precipitation modes – results in higher bioactivity of
the protein.
Bottleneck
Removal of polymer after precipitating the protein –
• Low concentrations of the polymer are removed in
subsequent procedures.
• Ultrfiltration or salt-induced phase separation can be
used.
 Precipitation of lipase by different precipitating
methods from cell culture filtrate

Precipitating Total activity Total protein Specific activity Recovery of

agent in ppt (U) ppt (mg) (U/ mg protein) enzyme (%)
Ammonium 4850 170 28.53 97
sulfate
Acetone 4600 119 38.65 92
Ethanol 3950 98 40.30 79
Isopropanol 4200 106 39.62 84
Acetic acid 3600 90 40.00 72
PEG 4000 4200 106 39.62 84
PEG 6000 3800 106 35.84 76
PEG 20000 3500 108 32.40 70
Initial total activity of the enzyme = 5000 U; protein = 710 mg